The Use of X-Ray and Nitrogen Mustard To Determine
the Mitotic and Intermitotic Times in Normal
*
and Malignant Rat Tissues
WILLIAM
R.
WIDNER,t
JOHN
(Los Alamos Scientofic Laboratory,
B.
STORER,
AND
University of California,
C.
C.
LUSHBAUGH
Los Alamos, New Mexico)
The relative importance of the times involved
in the various phases of cellular proliferation is bas
ic to problems of growth but has received little
attention until recently. Data on the quantitative
comparison of the rate of cellular proliferation of
mammalian tissues have also been meager. In a
phase to continue through their division in an ap
parently
norma! manner (4, @1, @4, @5).This
blockade causes a progressive fall in the mitotic
index (MI) (defined as the number of mitoses per
cell and for convenience expressed as a whole
previous
number
report
(16),
an
indirect
method
was
tosis
pre
sented for determining the time consumed in mi
tosis and in “resting―
between mitoses by cells of
mammalian
tissues. The data from this experi
ment indicated that the time spent in mitosis was
such an insignificant portion of the cell-doubling
time that the rate of proliferation of normal cells
has an inverse relationship
to the length of the
resting stage or interphase.
If this relationship
were found to apply also to neoplastic cells, chemo
therapy of neop!asia might be rendered more ef
fective by substances which would prolong the
resting
interfere
stage
of neoplastic
with
their
mitosis.
cells rather
than
only
No extensive
search
has as yet been made for substances
tion,
and
few
if any
are
known
with such ac
to exist.
The present study was undertaken (a) to extend
the data of the previous report on normal mouse
tissues to rat tissues, (b) to compare the time com
ponents of the mitotic process of neoplastic and
norma!
cells,
(c)
to
compare
quantitatively
allowing
times
those
10—i). The
already
rate
of fall
past
mid-pro
in the
mitotic
index is dependent upon the time required for the
cells already in mitosis to complete their division.
Therefore, by determining
the mitotic index of
tissues
at intervals
following
irradiation
until
a
mitotic index of zero is reached, the mitotic time
(MT) would be determined. In practice, however,
a mitotic index of zero is rarely attained, because
the initiation of mitosis by a few cells is not in
hibited by the lower doses of radiation and because
higher doses injure some dividing cells sufficiently
to cause a pronounced slowing or complete arrest
of mitosis. For this reason, it becomes necessary to
extrapolate the slope of the decline of the mitotic
index to zero. Since, with few exceptions, the mi
totic index does not immediately
begin to fall
following radiation, it is also necessary to extrapo
late the slope upward to intersect the line repre
senting the normal mitotic index. This is done by
drawing a line calculated by the sum of the least
the
rate of cellular proliferation in various normal and
neop!astic tissues, and (d) to determine whether or
not the radiomimetic
drug, methy!bis(@9—chloro
ethyl)amine
hydrochloride,
(HN@), a nitrogen
mustard, has the same effect as x-ray on mitoses
and whether it could be substituted
for x-ray in
this method of determination.
EXPERIMENTAL
while
METHOD
The method for determining the length of time
involved in mitosis depends upon the fact that
ionizing radiation prevents cells from entering mi
squares method to fit the experimental
points.
The time intercept between the point of inter
section of this line with the normal value and with
zero
represents
the
mitotic
time
(Chart
1).
Since high doses of radiation damage some cells
sufficiently to cause a slowing of division and low
doses of radiation do not prevent some cells from
entering mitosis, it is important to select a dose
intermediate
between these extremes, inasmuch
as both extremes in dosage would tend to give re
suits
showing
excessively
long
mitotic
times.
Several doses of radiation were therefore employed
in this study. The dose giving the shortest mitotic
time was considered to be the optimum for the
tissue in question, and comparisons
t This @*per
ispartofathesissubmittedto theUniversity particular
of New Mexico in partial fulfillment of the requirements for were made between tissues on the basis of time
the Ph.D. degree.
derived by employing this “optimum―
dose.
Received for publication August 7, 1951.
From the mitotic index (MI) and the mitotic
*
Work
done
under
the
auspices
of
the
ABC.
877
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878
Cancer Research
time (MT) expressed in minutes, the intermitotic
or resting time (IT), in minutes, may be calculated
from the formula IT = MT/MI
(18). This holds
only for stationary
cell populations
where the
number of cells produced is balanced by the num
ber removed or destroyed as in most normal adult
tissues. In tissues showing exponential
growth,
e.g., neoplasms,
the formula
becomes IT =
0.693 MT/?vII, the factor 0.693 being necessary
because the number of cells in the tissues is in
creasing exponentially with time (1@).
placing the thimble of the ionization chamber ad
jacent to the tissue in question. A dose of 800 r to
the skin had fallen to 797 r when measured at the
site of the tumor and to 54@ r at the site of the
jejunum.
Rats
receiving
the nitrogen
mustard
were given
doses of 0.8 or 4.0 mg/kg in physiological saline
into the tail vein. Since it is known that nitrogen
mustard reacts with the tissues in 5 minutes or
less (15), the time required
for this interaction
@1
-I
U)
-‘I
@
to
occur within the tissues is comparable to the time
required to deliver the x-ray dosages.
MATERIALS AND METHODS
The control animals were killed and autopsied
just
prior to sacrifice of the treated animals. AU
Five hundred and fifty male Sprague-Dawley
rats were used in these experiments. Before use, animals were sacrificed by crushing the skull, and
the animals were acclimatized to the conditions of the tissues were removed and fixed immediately.
In the groups treated with x-ray, animals were
the laboratory for 7—10days. For the determina
tions on neoplasms, the Walker rat carcinoma
@56 killed at 5-minute intervals for @5minutes from
the mid-point of irradiation, and the following tis
sues were taken in this order : femoral marrow,
MitotiC Time (27.5 mm.)
Jensen or Walker tumor, and jejunum. The ani
U)
Normal Mitotic ridix
mals treated with 0.8 mg of nitrogen mustard/kg
were killed at 5-minute intervals from the time of
80
injection during the first hour and at 10-minute
intervals during the second hour. The following
U)
tissues
were removed in this group : Jensen or
P
Walker
tumor, and jejunum. In the group receiv
6
Oburved fdl in M)totic
ir,dex (Jijunum, 400r)
ing
4
mg
of nitrogen mustard/kg
only sections of
4
—
Lir'S COIcI@OtId
by sum ot
jejunum were taken. These were obtained by kill
least squares method to
2 f,t •xp*-imentoIpoints
ing animals at 5-minute intervals for 50 minutes.
The bone marrow specimens were prepared from
o__
5
10
$5 •2
TIME- MINUTESAFTER M1O-PO)NTOFEXPOSURE
the femur by the method previously described
(16). The remaining tissues were fixed either in
CHART
1.—Method
of determining
length
of mitotic
time
from changes in the mitotic index following x-irradiation.
Carnoy's fluid or in 10 per cent formalin, and
paraffin sections 6 micra thick were prepared.
and the Jensen rat sarcoma were used. Transplan
With the exception of the bone marrow, which
tation of the tumors was made subcutaneously
in was stained with Wright's stain, all tissues were
the manner previously described (11). Seven to stained with Mayer's hematoxylin and counter
14 days after inoculation of the rats with one of stained with eosin or Fast green FCF.
The mitotic counts were done under an oil im
the tumors, the animals were treated with either
mersion lens. For the purposes of standardizing
x-ray or nitrogen mustard. They were then killed
the counting technic, cells were arbitrarily consid
at intervals following treatment, and the mitotic
indices of the tissues were determined at each of ered to be in mitosis only between the appearance
of recognizable elongation of the chromosomes in
the various time intervals.
prophase and the final separation into two daugh
Groups of rats were given doses of %00, 400, and
800 roentgens of x-ray in a single total-body ex ter cells. In the bone marrow, 1,000 cells per smear
in both the mye!ocytic and erythrocytic
series
posure (p.50 kvP, 15 ma, inherent filtration equiva
were counted, and the number of mitoses in each
lent to
mm. Al, @00r/min). During exposures,
was determined. The number of mitoses per 100
the rats were confined in a flat lucite cage so con
suitable adjacent
crypts of Lieberktthn
were
structed that shielding of one animal by the other
counted in the jejunum by the method of Dustin
was impossible. The radiation doses were measured
(7), after first determining the average number of
in air with a 800 r Victoreen integron chamber
cells per crypt by counting the total number of
placed in the center of the cage. Since tissues ab
sorb ionizing radiations,
determinations
of the cells in @,000crypts. In the tumors, the incidence
of mitoses per 650 cells was determined by the
dose delivered to the site of the tumor implants
Chalkley method (6).
and the jejunum were made by opening rats and
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
WIDNER
et aL—Determinaizon
The mean value and standard
error were cal
culated for the values obtained for each experi
mental
mean
were
point.
Mitotic
value by more
excluded
from
counts
than
the
differing
from the
two standard
deviations
results.
A minimum
of
eight animals per point was used for the x-ray
studies. The minimum number of animals was re
duced to 5 per point for the nitrogen mustard
study without reducing the reproducibility
of the
results. Because the average value for the normal
control animals was to be used in calculating the
mitotic and intermitotic times, in addition to the
mitotic index, it was desirable to have the value as
statistically
significant as practicable. Therefore,
an average of sixteen animals was used to deter
mine
@
@
the
control
IS
14
•\
-I
Control
t@12
00 I(
0
in8
@2@r
\.
@
@
@\4OO
%\
4
0.
600
2
r
@-
‘
•
RAT JEJUNUM
0
5
879
of Mi@oses
In the initial decline of the mitotic index follow
ing treatment,
the values for tissues of animals
treated with nitrogen mustard did not reach as
low levels as did the tissues of animals exposed to
x-rays. This is probably due to an increase in the
number of dividing cells damaged sufficiently to
value.
RESULTS
The mitotic index of each tissue showed a fairly
uniform decrease after x-radiation
or nitrogen
mustard injection. With the exception of the je
junum from the animals treated with nitrogen
mustard, this decrease became manifest 5—10mm
utes following treatment. The jejunum from ani
mals treated with nitrogen mustard showed no
rapid initial decrease as did the other tissues, but
there was instead a somewhat slower fall beginning
at 40 minutes. The changes in mitotic indices are
shown in Charts
@-7.It can be seen from these
graphs that in the animals treated with nitrogen
mustard there was a sharp initial drop in mitosis
in the tumors followed by a secondary increase in
the count and then a second slow fall extending to
C,)
-J
of Time
•0
10
15
20
25
TIME -MINUTES AFTER MID-POINT OF EXPOSURE
CHART @.—Changeain the mitotic index of the rat jejunum
at intervals followingvarious doses of x-radiation.
the end of the observation period. The determina
tions on x-ray-treated
animals were not carried on
for long enough time intervaLs to find out whether
a similar recovery and secondary fall were present.
SPiUTES ArTES MID-POINT
EXPOSURE
CHART3.—Changes in the mitotic index of the myeloblastic
and erythroblastic series in rat bone marrow following various
doses of x-radiation.
slow or stop mitosis. More abnormal mitotic fig
ures were seen in tissues from animals treated with
nitrogen mustard than from animals treated with
x-ray. These abnormalities consisted principally of
prophases showing “stickiness―
of chromosomes,
metaphases lacking spindle fibers, and “bridging―
of chromosomes in the late phases. Mitotic times
were calculated on the basis of the initial rapid de
chine in the mitotic index using the method as de
scribed above (Chart 1). The times derived by
using the various dosage levels are shown in
Table 1. The shortest mitotic time obtained for
each tissue is indicated by an asterisk in Table 1
and is listed separately in Table @.
These are con
sidered to be the most accurate determinations
for reasons discussed above. These times vary
only from @5to @7minutes; therefore, the actual
time spent in mitosis is probably fairly constant for
a wide variety of rat tissues including experimental
tumors. Furthermore,
this time is so brief that it
is of negligible importance
in the cell-doubling
time.
The intermitotic or resting times of cells of the
various tissues were calculated from the formula
IT = MT/MI
for the normal tissues and by the
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
880
@
Cancer
formula IT = 0.698 MT/MI
for the neoplasms.
These times are listed in Table 1. In Table
the
tissues are listed in the order of their relative rates
of proliferation as indicated by the intermitotic
Research
DISCUSSION
The use of x-radiation
to inhibit mitosis in
early prophase appears to be a relatively simple
in @it,o
method for the determination of how long
a time the cells of various tissues spend in cell di
vision. From the data obtained by this method,
6
@
I'7@
@t4.OmgHN2/Kg
it
I'
It
@,1
\
Control
:@
4
2
RAT
@
)
so
JEJUNUN
zo
qo
@o 60
70
80
90
tOO 110
30
40
TIME - MINUTES AFTER INJECTION
CHART
WALKER RATCARCIP4O-SARCOMA
256
6.—Changes
lowing treatment
in the
mitotic
index
of rat
jejunum
fol
with nitrogen mustard.
TIME- MINUTES AFTER MlD-P@P4T
OF EXPOSURE
CHART
cinoma
4.—Changes
in the
mitotic
index
of Walker
rat
car
@56following various doses of x-radiation.
0,
40
TINE
CHART
7.—Changes
MINUTES AFTER INJECTION
in the
mitotic
index
of Walker
rat
car
cinoma @56
and Jensen Sarcoma following treatment with ni
trogenmustard.
JENSEN
RAT SARCOMA
TlME-@NUTES
CHART 5.--C)1Rfl@5
AFTER MIO-P@NT
in the snitotic
OP EXPOSURE
index
of Jensen
rat sar
coma following various doses of x-radiation.
times. As might be expected, the two tumors show
the shortest resting time followed by the myelo
cytic series of the bone marrow and the jejunum,
which are both approximately
equal in their rates
of proliferation,
and finally by the erythrocytic
series of the bone marrow.
all tissues of the rat that were examined seem to
take essentially the same length of time to corn
plete cell division. H the early portion of prophase,
which cannot be measured accurately, is ignored,
mitosis of normal and malignant rat tissues re
quired
@*5.4
±0.9 minutes for completion. With
this figure established for the rat, it is possible to
substitute it as a constant in the formula for the
derivation of the length of the resting stage so that
the formula now reads : intermitotic
time (mm
utes) = @5.4/number of mitoses per one cell, or
IT = [email protected]/MI. Where a tissue is growing ex
ponentially,
the formula
is: IT = 0.698 (@$.4)/
MI or IT = 17.6/MI. Thus, in the case of the re
generating rat liver, where mitotic arrest could
not be produced by amounts of radiation that were
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
WIDNER
a al,—Deterininatwn
0.009 mitoses per cell. Using the equation for ex
ponential growth and solving for IT using this
mitotic index, the resting stage of the regenerat
ing hepatic cell after 46 hours was 1,955 minutes
or [email protected] in length. Similarly, the length of the
resting stage for cells of other tissues or tumors
can now be found for the rat by determining the
rnitotic index and solving the above formulae.
The term “rateof cellular proliferation― is used
commonly in oncology and in other studies of
P.
Knowlton
and
W.
R.
Widner,
unpublished
data,
1949.
TABLE
MIT0TIc
INDEX,
DETERMINED
MIT0TIc
TIME,
1
AND INTERMITOTIC
BY USING V..tRlous
881
of Mitoses
normal and abnormal growth, but it is rarely de
fined. While it is true that the rate of cellular pro
liferation is proportional to the mitotic index, the
mitotic index is actually a measure of the length
of the resting stage of cellular life and does not re
flect the speed of the mitotic process, which is ap
parently constant. Thus, the pathologist's rule of
thumb that the higher the mitotic index, the
greater the rate of cellular proliferation of the tis
sue is correct, but actually is another means of ex
pressing the concept that the shorter the resting
stage of cellular life, the greater the number of cells
in mitosis at any time. The numerous possible
applications and uses of this concept in problems
of cellular growth are obvious. For example, using
the mitotic indices of the regenerating rat hepatic
feasible,' it is possible to determine the length of
the resting stage by knowing the mitotic index at
any time. At 46 hours after partial hepatectomy,
the rnitotic index was 9.0 per thousand cells or
1 N.
of Time
Dosi@s OF X-RAY
TIME OF RAT TISSUES AS
OR NITROGEN
MUSTARD
Intermitotic
Mitotic time
Mitotic index
(13.7±1.1)10@
Treatment
Tissue
Jejunum
@00r x-ray
400 r x-ray
800 r x-ray
(minutes)
105.9
@75*
29.8
54.9
20.8
0.8 mg HN@/kg
4 .0 mg HN@/kg
Myelocytic series
of marrow
@00r x-ray
400 r x-ray
800 r x-ray
(13.2±0.6)10@
81.4
25 7*
29.0
Nudeated
@00r x-ray
400 r x-ray
800 r x-ray
( 6.5±0.4)1O@
42.5
Walker rat carcino
ma
@00r x-ray
400 r x-ray
800 r x-ray
0. 8 sag HNWkg
(25.2±1.6) 10'
Jensen rat sarcoma
@00r x-ray
400 r x-ray
800 r x-ray
(25.0±2.3)
red
blood cells
(31.9±2.0)10@
0.8 mg HN@kg
6
Indicates
times
considered
most
accurate
for
(10'
(32.0±1.9)10'
reasons
discussed
TABLE
in
the
time
(hours)
129.2
83.5*
86.4
66.9
25.3
39-5
32.4*
86.6
24.6*
105.4
87.7
61.0*
30-s
38.4
24.8*
42.6
14.2
15.4
11.4*
15.5
30@5
28.4
26.6*
62.7
14.2
18.2
12.3*
85.4
22.7
text.
2
C0MP..tRisoN OF TILE RATE OF PROLIFERATION OF MOUSE (16) AND RAT TISSUES
AS INDICATED BY ThEIR MIT0TIC INDICES AND INTERMITOTIC TIMES
Mouss
RAT
Inter
Inter
Mitotic
Mitotic
mitoticMitoticMitoticmitotictimeindextimetimeTissux(hours)(X1O')(minutes)(hours)Walkercarcinoma25.224.811Jensensarcoma25.026.612M@relceyticseries
index
time
(X1O')
(minutes)
Jejunum3.8
33Erythrocyticseries
9.335.8
28.9155
Ovary
Lymphnode
2.8
0.67
0.75
0.2229.5
21.1
28.2
30.2
14.499
Epidermis
Adrenal5.0
* Recent
experiments
indicate
this
value
should
be
4818.2
18.725.7
27.582
123
580*
670
10906.524.661
about
100
hours.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
88@
Cancer Research
cells, as published by Brues and Marble (f), and
solving the formula above, it is seen that the rest
ing time of the hepatic cells varies with the time
after hepatectomy.
At the end of Q4 hours when
the mitotic index was 0.0@13 per cell, the intermi
totic
@
time
was
13.8
hours;
at 48 hours:
mitotic
in
dex, 0.0097, and intermitotic time, 80.@ hours; and
at 7@ hours: mitotic index, 0.0063, and intermi
totic time, 46.6 hours. In the normal liver, where
one mitosis is seen among %0,000 cells, the resting
stage is by these calculations 8,466 hours or 353
days in length. Stimulants
to increased rate of
cellular proliferation appear to affect this change
by shortening the intermitotic time. Thus, the rat
bone marrow when stimulated by subcutaneous in
jections of turpentine undergoes myelocytic hyper
plasia, the mitotic index is increased, the time in
volved in visible mitosis remains
@5.7minutes,
and the resting stage of the myelocytes is decreased
from 3%.4 to @4.9hours.2
The application of the concept of the impor
tance of the resting stage in growth to problems in
oncology and chemotherapy
of malignant tumors
may be fruitful. For example, in a study of the
tion of prophase at the time of irradiation, but
most cells past the stage of breakdown in the nu
clear membrane complete division at a normal
rate over wide ranges of radiation dosages (4, @1,
@4, @5).The slight delay in the fall of the mitotic
index is most likely due to the fact that inhibition
of mitoses occurs at a stage slightly earlier than
the arbitrary limit set as the beginning of pro
phase for the purpose of counting. The mitotic
times measured by this method did not represent
the total time required for a cell to divide, but only
that time required for cells to complete division
from the stage of recognizable prophase. The time
required for the cells to go through early pro
phase to the point of recognizable elongation of
the chromosomes, therefore, could not be taken
into account. This time has been estimated van
ously to take 30 minutes to hours (3, 5, 14, 18,
19). Since the mitotic index also does not include
cells in this early part of prophase, the intermitotic
time
derived
from
the
division
of the
mitotic
time
by the mitotic index should be an accurate value,
because the error cancels out in the formula
IT = MT/MI.
The mitotic times obtained in this experiment
effect of urethan on Walker rat carcinoma
@56
compare closely with those obtained by other in
(11), Green and Lushbaugh concluded that ure
than inhibits cellular proliferation
in part by vestigators using different methods of determina
tion. A comparison of these times with those re
nutritional
debilitation of the animal and by in
ported by other investigators is made in Table 3.
creasing
the production
of nonproliferating
daugh
The majority of the times derived from tissue cul
ter cells. Restudying these data with the concepts
tures were made by direct observation of mitosis.
and formulae described here, it is found that the
resting stage of the carcinoma cells was 14.6 Part of the variation in the times is due to dif
ferences in the definition of the limits of mitosis.
hours in normal well fed animals but [email protected] hours
when growing in animals that had been depleted of Variable portions of prophase are included, and
there is poor agreement as to when telophase ends.
their proteins for S months. In urethan-treated
Only in the in vivo experiments with mice and rats
rats, force-fed in an attempt
to overcome the
in which x-ray was used as the mitotic inhibitor
starvation that accompanies this drug, the resting
is the same portion of mitosis being considered.
stage was @0.9hours in length. Thus, it would
Despite
these variations, it is obvious that the
seem from these observations
of changes in the
length of the resting stage that a possible mode of mitotic times are of similar magnitude for a wide
variety
of tissues
as determined
by many
different
action of urethan on this tumor was to prolong the
technics.
time between mitoses. Further study of the length
A comparison of the mitotic and intermitotic
of the resting stage of other types of tumors in the
rat and other animals and its modification by times of the various tissues studied in this and a
previous report (16) is given in Table
@.It is be
drugs would seem to be a potentially
profitable
lieved
that
the
mitotic
and
intermitotic
times for
avenue of approach
to the chemotherapy
of
the
rat
tissues
are
more
accurate
than
those
given
cancer.
for the mouse tissues, because animals were killed
The accuracy of this method for determining
the mitotic time must be considered. While it is at 5-minute rather than 10-minute intervals; thus,
possible that the rate of completion of mitosis by more points from which to calculate the times
were available.
In addition,
three
doses
of x-ray
cells exposed to x-radiation is slowed, giving un
were used rather than one, and the “optimum―
duly long values for the mitotic time, the evidence
of other investigators indicates that this is not so. dose was used in calculation of the mitotic and
mtermitotic
times
for the rat tissues.
The value
There may be a pronounced delay in the comple
for the mouse lymph node might be in error.
More recent experiments on mice have indicated
2 C. C. Lushbaugh,
unpublished
data,
1950.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
WIDNER
et aL—Determination
times.
In every
case
except
that
of the
jejunum from the rat receiving 4.0 mg/kg, the
mitotic times derived were longer than those de
nived by using x-ray. The differences in fall of the
mitotic index were probably caused by a difference
in the mode of action on mitosis of the two agents.
It has been suggested by other investigators that
nitrogen mustard exerts its inhibitory effect on
the mitotie cycle in the premitotic stage (1, 8, 10,
@0, @),which would delay the fall of the mitotic
count following treatment. Such a delay was found
in the jejunum and tumors of the rats treated with
nitrogen mustard. The tumors also showed an
early rapid fall similar to that produced by x-ray
as well as the delayed fall. This is believed to in
dicate a second point of mitotic arrest by the ni
trogen
mustard.
Despite
dine of the mitotic
3 J. B. Storer,
the
similarity
of the
index for the tumors
unpublished
data,
888
of Miloses
with nitrogen mustard and the tissues treated
with x-ray, the times derived by the use of the
nitrogen mustard were longer than those derived
by using x-ray because of the increased number of
damaged cells in which mitosis was stopped. This
“colchicine-like―effect has been described by
Fniedenwald et al. (9) Because of this increased
mitotic time, the intermitotic times for the tumors
of animals treated with nitrogen mustard were
longer than for those of animals treated with x-ray.
that the intermitotic
time of lymphocytes is ac
tually closer to 100 hours.3
Nitrogen mustard is not a satisfactory substi
tute for x-ray in this method of determining
mitotic
of Time
SUMMARY AND CONCLUSIONS
1. By determining the rate of decline of the
mitotic indices of the jejunum, bone marrow,
Jensen sarcoma, and Walker rat carcinoma
@56
of rats immediately following exposure to x-radia
tion, iVwas found possible to determine thelength of
time spent by the cells in mitosis and in the resting
stage. Comparison of the results of this experi
ment with those of others revealed a close agree
ment between the mitotic times for a wide variety
of tissues, derived by several different methods.
de
treated
1951.
@. From
TABLE
these
data
it
would
appear
that
the
3
Co@&PARIsoNOF TIlE TIME REQUIREDFOR MrroslS BY CELls OF VARIOUSTIssUEs
Mitotic
Animal
Chick
“Embryonic―
Tissue culture
Fibroblasts
a
a
Cat
a
a
a
a
a
Connective Tissue
a
a
a
a
a
a
(38°—39°
C.)
a
a
(34°36° C.)
“
a
a
a
a
a
.25
15-30
25—45
21—29
35—50
70
In rivo using radium to in
Walker rat carcinoma
In vise using x-ray to in
hibit mitosis
24.8
Jensen rat sarcoma
Marrow, myeloid series
Marrow, erythroid series
Same as above
26.6
hibit mitosis
Jejunum
Jejunum
Marrow, erythroid series
Marrow,
myeloid series
Ovary
Lymph node
Epidermis
Adrenal
Times
derived
calculated
from tissue
by
us
from
other
culture
authors'
a
a
a
a
a
mm
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
mm
a
a
a
were from direct
data
by
the
Lambert and Hanes (1913)
cited by Lewis and Lewis
(19)
Ibid.
Lambert (1918) cited by
Lewis and Lewis (19)
Ibid.
Buschke, Friedenwald, and
Ibid.
Mottram,
(21)
Scott, and Russ
Table 2, this paper
a
a
a
a
a
a
a
a
25.7
24.6
27.5
23.9t
29.5t
35.st
21.1
28.2
80 . 2t
14.4t
observation
method
Wright (27)
Lewisand Lewis(19)
Fleischmann (3)
84 3*
88*
Jensen rat sarcoma
a
a
a
Author
Levi (18)
Simon-Reliss and Spear (23)
Juul and Kemp (14)
Willmer (26)
34 (av.)
32 (av.)
Its vwo using ether to in
hibit mitosis
Same as above
a
* Times
a
a
a
Cornea
Note:
a
a
18—20(av.)
20 (av.)
15—40
Heart
Mesenchyme
Rat
Mouse
time
(minutes)
Method
Tissue
described
or by repeated
in
this
Knowlton
and Widner (16)
a
a
a
a
a
a
“
a
a
a
a
a
a
a
a
photographs.
paper.
t Times derivedby Knowltonand Widner(16) by modificationof the methoddescribedin this paper.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
884
time
Cancer Research
required
for any
cell of the
rat
to produce
two
daughter cells following breakdown of the nuclear
membrane in early prophase is constant and meas
ures approximately
@5.4minutes. It would also
appear
mitotic
noma 256. Cancer Research, 9: 199—209,1949.
12. HOFFMAN,J. G. Theory of the Mitotic Index and Its Ap
plication to Tissue Growth Measurement. Bull. Math.
premitotic
of a tissue
Study of the Mode of Inhibition of Cellular Proliferation
pendent upon the length of the interphase or rest
ing stage and that changes in rate of cellular
proliferation are the result of changes in the length
of the resting stage.
3. Itwasnotfound
possible to substitute nitrogen
mustard for x-radiation as a means of producing
mitotic arrest in this method of determining mi
totic time, because nitrogen mustard apparently
early
index
Sulfides. Science, 103:409—15,486, 1940.
11. GREEN, J. W., and LUSHBAUGH, C. C. Histopathologic
by Urethane: Effect of Urethane on Walker Rat Card
an
the
F. S. The Biological Actions and
of the @9-ch1orethyl Amines and
is de
causes
that
10. Gnaur@, A., and Pmups,
Therapeutic
Applications
arrest
as
well
as
an
arrest in early prophase, while also having a
coichicine-like effect later in mitosis. These dif
ferences
in mode
of action
of nitrogen
mustard
upon cell division lead to unduly prolonged mitotic
times as derived by this method.
ACKNOWLEDGMENTS
F. Castetter
are gratefully
acknowledged.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1951 American Association for Cancer Research.
Various
in vigro.
The Use of X-Ray and Nitrogen Mustard To Determine the
Mitotic and Intermitotic Times in Normal and Malignant Rat
Tissues
William R. Widner, John B. Storer and C. C. Lushbaugh
Cancer Res 1951;11:877-884.
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